NK cells are the first line of defense against tumor cells. We recently developed CAR NK cells for the treatment of multiple myeloma (MM), which demonstrates improved anti-MM activity. However, challenges still exist for NK cell- based cancer immunotherapy: some tumor cells are resistant to cytotoxicity of NK cells that have a short life span after infusion into patients. Thus, further understanding of NK cells will be critical to better harness this population for immunotherapy. Our data recently published in Nature Immunology show that an important transcription factor, XBP1s, encoded by an unconventionally spliced mRNA of X-box binding protein (XBP1), positively regulates NK cell survival and effector functions. XBP1s regulates NK cell cytotoxicity and GZMB gene expression via direct promoter binding. XBP1s physically interacts with T-BET, a master regulator in NK cells, suggesting that XBP1s can recruit T-BET to the promoters of target genes. This discovery fills a current gap in the field, as T-BET is known to positively regulate the expression of GZMB despite lacking direct T-BET binding sites on the GZMB promoter. Moreover, IL-15, one of the most important cytokines regulating NK cell survival, development, and effector functions, activates AKT signaling to increase stability of XBP1s protein via deubiquitination, leading to XBP1s nuclear accumulation. Consistent with these human data, conditional knockout of Xbp1 in mice shows a decreased number of NK cells and impaired NK cell anti-tumor activity. We believe that we have identified a novel IL-15-AKT- XBP1s signaling pathway that plays key roles in regulating multiple aspects of NK cell biology. Thus, our overall hypothesis is that this IL-15-AKT-XBP1s signaling pathway newly identified by our group positively regulates NK cell survival and effector functions and can be utilized to improve NK cell or CAR NK cell- based cancer immunotherapy. We propose mechanistic studies regarding how XBP1s regulates NK cell survival and effector functions. We also propose combinational therapies of IL-15/IL-15R? with CS1-CAR NK cells or with B-I09, a novel drug targeting XBP1 splicing to XBP1s that has strong activity in treating various cancers including MM. IL-15/IL-15R? can selectively stabilize the XBP1s protein and prevent B-I09-mediated downregulation of XBP1s and the associated inhibition of functions in NK cells. We will engineer CS1-CAR NK cells to express IL- 15/IL-15R? or XBP1s to have enhanced in vivo persistence of these cells for continuous tumor eradication. Three Aims are proposed. Aim 1: Study the mechanism and functional consequences of enhanced XBP1s protein expression levels via IL-15-AKT signaling in NK cells. Aim 2: Study whether and/or how XBP1s regulates NK cell survival, proliferation, and trafficking. Aim 3: Study the role of XBP1s in combination therapies of IL-15/IL-15R? with a novel drug targeting XPB1s, B-I09, and/or with CS1-CAR NK cells for the treatment of MM. Our project is significant and timely as IL-15 and IL-15/IL-15R? are top agents for cancer immunotherapy and are being actively tested in the clinic. Our studies will lead to new insights into NK cell biology, improve outcomes for NK cell-based immunotherapy, and facilitate developing innovative cancer immunotherapeutics.